In addition to their bioenergetic function, mitochondria are critical regulators of Ca signaling in neurons. Mitochondria efficiently buffer Ca2+ influx during excitation and limit the amplitude of the cytosolic Ca2+ concentration ([Ca2+]i) increase. Rapid Ca2+ uptake is followed by a slower Ca release from mitochondria, completing stimulus-induced mitochondrial Ca2+ cycle. By shaping [Ca2+]i response, mitochondria can modulate numerous Ca2+-dependent neuronal functions. At the same time, impairment of mitochondrial Ca2+ transport is the key factor leading to neuronal damage in stroke and in a number of neurodegenerative disorders. Despite significant progress, many questions remain about the spatiotemporal organization, function and modulation of mitochondrial Ca2+ cycling in neurons and, specifically, about the mechanisms regulating the transition from physiology to pathophysiology. Our overall hypothesis is that mitochondrial Ca2+ cycling controls diverse neuronal functions and that the decision between physiological and pathological outcomes is influenced by reversible phosphorylation of mitochondrial proteins. We will initially focus on two physiological processes, transmitter release (Aim 1) and activation of transcription (Aim 2), by studying spatiotemporal organization and the role of mitochondrial Ca2+ transport in two morphologically and functionally distinct cellular compartments, presynaptic boutons and the cell soma, respectively. We will then investigate how protein kinase A and protein phosphatase 2A modulate mitochondrial Ca2+ signaling and Ca2+-dependent processes, such as neurotransmission, transcription activation and excitotoxicity (Aim 3). Both enzymes are targeted to the outer mitochondrial membrane (OMM), but exhibit opposite effects on cell survival, and are predicted to differentially influence mitochondria-dependent functions. The proposed studies will advance our understanding of how mitochondrial Ca2+ transporters interplay with protein kinases and phosphatases in neurons to trigger a specific physiological or pathological response.